Effects of a low, medium, and high-intensity aquatic physiotherapy protocol on functional and biochemical parameters in individuals with knee osteoarthritis: protocol for a crossover randomized controlled trial

Background Knee osteoarthritis affects the performance of daily activities, independence, and quality of life. The etiopathogenesis of this condition considers the mechanisms of activation of metalloproteinase and reactive oxygen species production pathways. Metalloproteinases-3 (MMP-3) and Glutathione Peroxidase (GPx) may be responsible for cartilage destruction. Aquatic physiotherapy promotes a positive impact on the clinical picture of osteoarthritis, and this study presents an intervention protocol that aims to evaluate the effects of a single session of different aquatic physiotherapy modalities on the biochemical and functional behavior of patients with knee osteoarthritis. Methods This will be a crossover randomized controlled trial in which 15 individuals will be submitted to three aquatic physiotherapy modalities with a minimum 15-day wash-out period in patients over 50 years old and diagnosed with OA in at least one knee, presence of pain and at least one functional dysfunction for at least 6 months, absence of physical limitation that prevents the exercise protocol from being performed, Kellgren and Lawrence ranking between I and IV, walk independently and without auxiliary device. Variations in the concentrations of MMP-3 and GPx in peripheral blood, pain, edema, and flexibility resulting from the three aquatic physiotherapeutic interventions will be evaluated both pre- and immediate post-intervention. The reference group will be submitted to the same aquatic physiotherapy protocols, however, only the biochemical parameters and the self-perception questionnaires will be evaluated. Registration ClinicalTrials.gov ( NCT05610696, 18/01/2023).


Onu Ilie
, University of Medicine and 2.

Introduction
Osteoarthritis is the most prevalent rheumatologic joint dysfunction in the world, the knee joint can be considered the most affected segment and is due to local and systemic risk factors. 1 Knee osteoarthritis affects its functionality, mainly the performance of activities of daily living such as locomotion or even the transfers necessary to maintain independence and quality of life. 2,3The evolution is generally slow and may evolve in stages or reflect as a progressive evolution over time, resulting in a worsening of the severity of the symptoms and disease, 4 which also has an important economic and social impact. 5According to the data from vizhub.healthdata.org,Brazil is in the 15 th position in the distribution ranking of osteoarthritis cases, and a prevalence of 3.42 cases per 100,000 inhabitants is estimated, with an incidence of 181.47 new cases.
Current understanding suggests that inflammatory processes are closely implicated in the pathogenesis of osteoarthritis and, in this sense, the articular cartilage, the target of degenerative processes, is modified in its composition and structure. 69][10][11] Many MMPs families have been reported to induce articular cartilage degradation. 7,12The metalloproteinase-3 (MMP-3) can be considered the great responsible for the destruction of cartilage resulting from the degradation of several types of collagens, 12 and this same enzyme was found in the plasma shortly after articular injuries, possibly due to the inflammatory activation process of the synovium, similarly, the plasma levels of MMP-3 are shown to be predictors of articular narrowing in patients with osteoarthritis. 13[16] Treatment strategies for osteoarthritis are described as evidence-based practices along with guidelines that indicate consensus of important organizations dedicated to the study of musculoskeletal conditions.Most study bases are focused on knee articulation, and, in this sense, the American College of Rheumatology (ACR), 17 The European Alliance of Associations for Rheumatism (EULAR), 18 and the Osteoarthritis Research Society International (OARSI) 19 reinforce that the practice of exercises and physiotherapy (both on the ground and in the liquid environment) are an important therapeutic arsenal for knee osteoarthritis.Although the evidence regarding the efficacy and safety of exercise is well elucidated, there is still a need to clarify the intensity levels of this activity safely.In this regard, the type of exercise and the various parameters concerning its intensity are part of the challenge for physiotherapists dedicated to rehabilitating rheumatic diseases.
It is currently well established that aquatic physiotherapy promotes a positive impact on the improvement of the clinical and functional status of patients with knee osteoarthritis, being considered an effective and safe modality once the immersion in heated water reduces overload and joint pain in addition to improving functional capacity and quality of life. 20This treatment modality also promotes the improvement of functional capacity and emotional well-being. 21mplementing aquatic physiotherapy for knee osteoarthritis requires considering how immersion affects exercise through forces like drag and turbulence.Thus, treatment plans should incorporate hydrostatic and hydrodynamic principles, physiological effects of immersion, and be based on individual assessments and evidence-based practices, 22 these factors confer upon aquatic physiotherapy in patients with knee osteoarthritis, a treatment strategy with fewer side effects when compared to other modalities. 235][26] However, it is still necessary to understand the impact of immersion and of different modalities of aquatic physiotherapy on the biochemical behavior of articular cartilage and correlate them with functional variables.It has recently been possible to understand the effects of an aquatic exercise program on the degenerative modifications in articular cartilage using an experimental animal model of knee osteoarthritis with an alteration in the expression of MMPs 27 and greater integrity and orientation of collagen fibers. 28VISED Amendments from Version 3 In this new version of the article, we have made some modifications to certain references and primarily revised the introduction section and the study limitations.
Any further responses from the reviewers can be found at the end of the article Results like these indicate the possible cellular and molecular mechanisms that justify the positive results from aquatic physiotherapy in knee osteoarthritis and reinforce the need for further studies in this area.In this context, the analysis of markers such as oxidative stress and MMPs constitutes a relevant topic of study.
Previous studies in a systematic review demonstrated that aquatic physiotherapy positively affects clinical, functional, and quality of life variables in individuals with osteoarthritis. 23,29Similarly, it has recently been evidenced that aquatic physiotherapy positively affects pain, physical function, knee extension muscle strength, and walking ability. 23,30hese studies are related to heterogeneous protocols in intensities, frequency, total treatment time, and a single type of program.
Although this intervention has been shown to be safe, there is a lack of data evidencing its effects on biochemical variables, especially with a comparative analysis between pre-and post-intervention times, making it relevant to provide equally safe physiotherapist protocols of varying intensities.Nevertheless, understanding the central mechanism responsible for functional improvement, including the impact of aquatic physiotherapy on biochemical variables and its correlation with articular collagen degradation, will undoubtedly contribute to enhancing this type of intervention as a treatment strategy for knee osteoarthritis through evidence-based practices.These factors confer upon aquatic physiotherapy in patients with knee osteoarthritis a treatment strategy with fewer side effects than other modalities.
Therefore, the protocol proposed in this study presents different modalities of intervention in aquatic physiotherapy, this being a single session, considering low, medium, and high-intensity strategies, seeking to evaluate its effects on the biochemical and functional behavior of patients with knee osteoarthritis.It aims to identify possible variations in the concentration of the MMP-3 subfamily and the oxidative stress marker Glutathione Peroxidase (GPx) in peripheral blood, assess pain, edema, and flexibility in patients with knee osteoarthritis and in response to different aquatic physiotherapy interventions.Therefore, understanding the appropriate exercise intensity, coupled with understanding its effects on biochemical mediators directly involved in the genesis of osteoarthritis, can provide confidence for the prescription and execution of therapeutic exercises under immersion in aquatic physiotherapy treatment.These strategies may increase treatment adherence, prevent symptom exacerbation, minimize adverse effects, and provide greater therapeutic safety.

Trial design
This is an interventional transversal study, randomized clinical trial, crossover type with three arms: low, medium, and high-intensity aquatic physiotherapy.This clinical intervention protocol follows the recommendation from the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT). 31,32The study sample, defined according to criteria, will be submitted to a single session of each one of the following modalities of aquatic physiotherapy: (a) low-intensity, (b) medium-intensity, and (c) high-intensity.Among each modality, a minimum of a 15-day wash-out period will be respected, in accordance with Figure 1.
For this study, 15 participants will be recruited.The sample size calculation indicated a non-centrality parameter (δ) of 3.8, suggesting a moderate effect on biomarker variations.The critical t-value was 2.10, with degrees of freedom (df) of 16.143, resulting in a total sample size of 15 participants and an actual power of 0.95.The effect size calculation was based on means and standard deviations, and for our main dependent variables, these were reported by previous investigations: Germanou 33 through the antioxidant levels and Nascimento 34 through the MMP-2 and MMP-9 analysis between the control and intervention group, in this case, it is represented by patients with knee osteoarthritis.
The assessment parameters used in each variable will consist of instruments that allow for the assessment of the variations in the MMP-3 concentration, oxidative stress, pain, edema, and flexibility.In addition, a self-perception questionnaire, developed by the authors, will be applied to assess self-perception.It consists of a Likert scale where the patient must classify their perception of well-being between zero and 10, where zero represents the worst feeling of well-being and 10 represents the best feeling of well-being.The data collection form, informed consent form and patient identification form can be found as Extended data. 32The trial has been registered on ClinicalTrials.gov(NCT05610696, 18/01/2023).

Study setting
The study population corresponds to people with knee osteoarthritis.The population will be recruited from physiotherapy services from University Feevale.The protocol will be performed in the facilities of the Aquatic Physiotherapy sector.The swimming pool has accessibility via stairs and a ramp with appropriate handrail support.Heating is provided by an electronic boiler fed with liquefied petroleum gas that guarantees minimum temperature variations (33°C and 34°C) measured with a submerged mercury column thermometer.The swimming pool depth varies between 100 and 150 cm, transitioned by means of a ramp; the entire swimming pool is lined with non-slip tiles.The swimming pool also has two hydromassage spaces on one of the borders.Each one is equipped with hydromassage jets, one in the trunk support region and two on the sides of this area.

Eligibility criteria
The following eligibility criteria will be considered: being over 50 years old and diagnosed with osteoarthritis in at least one knee according to the American College of Rheumatology 35 criteria, referring to pain and at least one dysfunction for at least six months, absence of physical limitation that prevents the performance of the exercises protocol, have Kellgren and Lawrence classification between III and IV 36 and independent gait.Subjects who present autoimmune inflammatory conditions, use of psychotropic, immunosuppressant, or steroids drugs, consumption of antioxidants (vitamin E, statins, or uric acid reducers), alcohol and tobacco, progressive ankylosis states, the coexistence of neurological lesions, infections or skin diseases that contraindicate swimming pool immersion, surgical procedures on the knee joint in the last 12 months (excluding meniscectomy or arthroscopy), lower limb articular prosthesis, presence of inflammatory or infectious disease, uncontrolled systemic arterial hypertension, uncontrolled diabetes, chronic obstructive pulmonary disease and history of ischemic heart disease, self-reported aquatic phobia, and chlorine allergy will be excluded from the study.For control purposes, a reference group will be constituted with age matching and exclusion criteria identical to the experimental group.

Outcomes
Initially, all participants will be interviewed and evaluated in relation to the degree of osteoarthritis and functionality.Information regarding the patient data will be confirmed via interview, considering data such as complete name, date of birth, age, affected knee(s), duration of pain, arterial blood pressure, list of medications in use, consumption of tobacco or alcohol, associated diseases, if they are undergoing physiotherapy treatment or regular physical activity.
The classification regarding the degree of osteoarthritis will be performed by analyzing the radiograph of the affected knee according to the method of Kellgren and Lawrence.The radiographs will be analyzed, the degree of osteoarthritis will be identified with the negatoscope support, and the images in the digital form will be displayed on a computer screen, considering the classification expressed in degrees ranging from zero to IV.Subsequently, the identification of functionality will be assessed according to the Brazilian version of the Knee Injury and Osteoarthritis Outcome Score (KOOS).The KOOS questionnaire was developed in the 1990s as an instrument to evaluate the patient's perception of their knee and associated problems.Since its first publication in 1998, the psychometric properties of KOOS were evaluated in a systemic review of 37 articles demonstrating adequate validity in its use for the study population. 37garding the pre-and post-intervention assessment instruments, all participants will have the following parameters evaluated: concentration of MMP-3 and GPx, pain, edema, flexibility, and self-perception of the general well-being.In addition, it should be noted that in the 24-hour-follow-up, only the biochemical concentrations and the self-perception questionnaire will be reevaluated.
The biochemical variables will be measured in peripheral blood from the concentrations of MMP-3 and GPx.The levels of MMPs will be measured based on the Enzyme-Linked Immunosorbent Assay (ELISA) methodology, which is based on the antigen-antibody interaction and will be performed according to the kit manufacturer's instructions.MMP-3, a constituent of the stromelysin family that is an important mediator responsible for the degradation of collagen type II, IX, and XI, 12,13 will be considered for this analysis.Oxidative stress will be measured by analyzing the peripheral blood from percutaneous punction.The sample collection time will be performed according to the study by Nascimento 34 and will be in accordance with the following times: T0 (immediately before the intervention), T1 (immediately after the intervention), and T2 (24 hours after the intervention).
The assessment of the degree of pain will be performed using the visual analogue pain scale, 38 from a scale graduated from zero to 10, in which zero corresponds to cases without pain and 10 corresponds to the worst imaginable pain.
The assessment of edema will be performed based on the measurement of the circumference of the affected knee(s).The measure will be performed with the patient relaxed in dorsal decubitus and the circumference will be determined with the knee in extension, based on the proximal distance of 1 cm from the base of the patella. 39For that, the same measuring tape will be used for all measurements.
The flexibility will be assessed by measuring the amplitude of the active and passive movement for the knee flexion and, for that, a conventional goniometer will be used.The goniometer will be used because it has a low cost, is easy to handle, allows the assessments to be performed quickly, and the measurements and movement amplitude for the knee joint are valid and reliable. 40For the assessment, the knee will be free of clothing, and the patient will be positioned in dorsal decubitus with the hip articulation at 90°of flexion.The fixed arm of the goniometer will be positioned parallel to the thigh with an orientation towards the greater trochanter of the femur and the mobile arm will be parallel to the lateral face of the fibula, directed towards the lateral malleolus.The movement reading will be performed while maintaining the final possible position of the arc of movement where the highest reading degree represents the assessed measurement. 41ll procedures will be performed by a single physiotherapist.

Interventions
The subjects of this study will be submitted to different interventions of aquatic physiotherapy systematized as follows: program A (low-intensity protocol), program B (medium-intensity protocol), and program C (high-intensity protocol).
The access to the immersion will be from a ladder, and a period of 5 minutes will be allowed for acclimatization to the liquid environment.Considering that the intervention proposals aim to provide different levels of articular overload, the speed and time of execution will be gradually variable.
Program A will use the principles of Ai Chi ® as this model is defined as a strategy of low intensity and consists of a sequence of slow movements. 42For that, a sequence of adapted exercises 43 will be performed, according to Table 1.
As a starting position to perform the exercises, the participants must remain in the semi-flexion position of their knees so that the water line must remain at shoulder height throughout the procedure.All the exercises will be performed according to the established time, and a 15-second rest time will be added between each exercise or series.As a manner of manipulating the intensity of the exercise at low intensity, the movement speed will be standardized according to the subjective sensation of effort according to Borg 6-20, 44 with zones of interest from 6 (extremely light) to 9 (very light).In this intervention modality, we will seek to understand the impact that the physiological effects of immersion in heated water pose on the studied variables.
Program B will consist of a sequence of exercises adapted from previous works 45,46 composed of warm-up, fortification, and cool-down activities.Aquatic steppes platforms will be used to ensure that all participants, when performing the exercises in orthostatism, will maintain the waterline level approximately at the level of the xiphoid appendix.For this program, the exercises will be performed at a speed that guarantees a comfortable water level for the activity, being the effort zones of interest from 10 (fairly light) to 14 (somewhat hard).The warm-up sequence consists of four different active moves performed at maximum movement amplitude and contributes to neuromuscular activation.
After the warm-up, the participants will be submitted to the fortification program, which consists of a sequence of seven exercises aiming to promote muscular activation.The participants will be encouraged to perform the movement at a comfortable speed with the maximum possible movement amplitude.During sitting exercises, the need to maintain the lumbar spine in a neutral position will be emphasized in order to avoid overloading this segment.
To finish program B, as a way to calm down, a sequence of three activities will be performed, including a period in the hydromassage area, according to Table 2.
In program C (high-intensity protocol), the same sequence of exercises stipulated for program B will be respected, however, the quantity of work will be modified based on the variables time and speed of the performance of the movement. 47Fortification activities will be performed for 6 minutes, and the speed must be adjusted in order to stimulate the performance of the movement for the subjective sensation compatible with the Borg's zone of interest 15 (hard) to Hip flexion (single leg support) concomitant with shoulder extension followed by hip extension (bipedal support) concomitant with shoulder flexion.

minutes, 2Â2 minutes for each articulation
Cross the forearms in front of the umbilical scar, followed by the horizontal flexion together with the movement of crossing one lower limb over the other.

minute
Sitting and standing up.

minute
Skipping -running on the same place.
Total warm-up time: approximately 10 minutes Fortification

minutes
In orthostatism with unipedal support of the lower limb contralateral to the moved segment, perform hip flexion and extension.
The knee must remain in complete extension and the ankle in dorsiflexion.The lumbar spine must remain in a neutral position.

minutes
In orthostatism, perform hip abduction and adduction.The knee must remain in complete extension with the ankle in dorsiflexion.The movement must contemplate the maximum amplitude with the maintenance of the pelvis and lumbar spine in a static position.

minutes
In orthostatism, perform the movement of knee flexion and extension.The movement must contemplate the maximum flexion and extension with the maintenance of the lumbar spine in a neutral position.

minutes
In orthostatism, perform the wide movement of hip extension and knee flexion followed by hip flexion and knee extension (kick movement).

minutes
With the help of floats, perform the bicycle movement (alternating knee and hip flexion) aiming at the anterior displacement.

minutes
Patient in a supine position with the help of floats, perform the backstroke leg kick aiming at the cranial displacement.

minutes
In orthostasis, perform the slow walking movement, simulating a Nordic walk, sliding the feet on the bottom of the pool, however, without moving.
Program B fortification program time: approximately 21 minutes.

seconds on each side
Active stretching of the gluteus maximus.

seconds on each side
Active stretching of the quadriceps.

seconds on each side
Active stretching of the hamstring.
18 (very hard).In the same way, walking activity will be replaced by running activity which amounts to an approximate time of 39 minutes.
The reference group will be submitted to the same aquatic physiotherapy protocols, however, only the biochemical parameters and the self-perception questionnaires will be evaluated.

Recruitment
The population will be recruited from the physiotherapy services of Universities.The study will also be disseminated in print and digital media, social networks of the researcher, the university, the academic directory of health courses, the Medical Leagues and institutional pages.

Assignment of interventions
Allocation: Participants will be allocated randomly to establish the sequence within each group, following a computergenerated randomization schedule from randomize.com,as exemplified by the flowchart in Figure 1.
Blinding: This study is classified as single-blinded, indicating that the researchers responsible for conducting the assessment instruments and laboratory analysis procedures will be distinct from those administering the intervention protocols.

Data collection, management, and analysis
Data collection methods: The interviews, evaluation of radiological images, application of instruments and measurements of pain, edema, and flexibility will be carried out only by a single qualified, trained physiotherapist, with familiarity with the instruments and with adequate training.Blood sample collection will be performed by a nursing professional familiar with this procedure.Laboratory analyzes will be performed by another researcher qualified biomedical pharmacist.The application of the intervention protocols will be carried out by two physiotherapists trained in aquatic physiotherapy and who will not participate in the interviews, radiological analysis and evaluation of edema, pain and flexibility.
Data management: All data will be entered electronically via Microsoft Excel.The study's digital forms will be inserted and received in a cloud file with access only to the researcher.Participant files will be stored in numerical order and stored in storage for a period of 3 years after completion of the study.Modifications to data recorded in the database will be documented through the data breach system associated with your user identification code and password.Access to study data will be restricted.All reports will be prepared in such a way that no subject can be identified.

Statistical analysis
The resulting data will be tabulated, considering the absolute values and their respective standards.Information regarding the main outcome corresponding to the variations in the concentrations of MMP-3 and GPx in peripheral blood due to three different aquatic physiotherapy interventions will be comparatively evaluated among the distinct interventions and among the different times (in pre-and post-intervention moments).Active stretching of the gastrocnemius.

minutes
Immersion in the whirlpool.
Total cool down time: approximately 8 minutes.
The information on the outcome regarding pain, edema, and flexibility will be comparatively assessed among the different types of intervention.Statistical analyses will be performed in the SPSS software and will be considered as a significance level when p>0.05 through the repeated measures ANOVA statistical test.

Data monitoring
We perceive the establishment of a Data Monitoring Committee as unnecessary due to the brief duration of this study and the established safety levels associated with aquatic physiotherapy for patients diagnosed with knee osteoarthritis.

Harms
In our study, an adverse event will be defined as any unfavorable functional occurrence in an individual consistently linked to exercise intensity.Adverse events will be documented after the subject provides consent and enrollment in the study.Researchers will evaluate the connection between an event and the exercise intensity of the study protocol, based on a temporal relationship, in addition to assessing whether the event is unexpected or unexplained.The study will meticulously track the subsequent protocol-associated adverse effects: heightened pain, inflammation, and diminished flexibility immediately post-intervention and at the 24-hour mark through patient examination.For any circumstance that may necessitate urgent medical support, the emergency medical service of the Aquatic Physiotherapy department will be activated to ensure the requisite and appropriate assistance.

Auditing
Regarding this matter, we find it unnecessary due to the nature of our single-center study, which involves a limited sample size and employs a therapeutic approach that has been well-established as safe.

Ethics approval and consent
This research was approved by the Research Ethics Committee of Federal University of Health Sciences in Porto Alegre in 15/05/2020 founder the number 4.030.865and Presentation Certified for Ethical Appreciation (CAAE) number 29447220.5.0000.5345.Patients will be invited to participate in the study voluntarily, and they must consent to their participation by formalizing the Free and Informed Written Consent according to established by resolution number 466 from the National Health Council. 48The study is planned to take place at University Feevale, and ethical approval has been obtained from the Research Ethics Committee of the Federal University of Health Sciences in Porto Alegre.This choice of institution for ethical approval is due to a strategic partnership between the two universities for this research endeavour.The partnership was established because our research advisor is affiliated with the Department of Physical Therapy at the Federal University of Health Sciences, making this institution the natural choice for ethical oversight.This partnership ensures that both institutions are jointly responsible for the successful execution of the study and ethical compliance is maintained throughout.The University Feevale is a co-participating institution in the research and, as such, declares its compliance with Resolution CNS 466/2012, which pertains to Brazilian regulations for research involving human subjects.

Protocol amendments
While the prescribed protocol has been deemed safe, having undergone pilot interventions within the aquatic physiotherapy department of the university, any modifications that might impact the study's execution will necessitate renewed consideration by the Ethics Committee.

Confidentiality
Participant study data will not be disclosed beyond the study without explicit written consent from the participants.This confidentiality is upheld through the previously approved Informed Consent Form.

Access to data
Sole access to the datasets will be granted to the Principal Researcher.Project data shall be securely stored within password-protected files.To ensure privacy, data shared with project team members will be stripped of any participantidentifying information.

Ancillary and post-trial care
The responsible researcher will promptly provide comprehensive support to research participants concerning potential complications and resulting implications.Similarly, considering the research's conduct within an accredited aquatic physiotherapy service, the service will consistently uphold any requisite therapeutic follow-ups after the study.

Dissemination
The study will be disseminated in printed and digital media, on the researcher, university, and the academic directory of health courses social media, in the Medicine Leagues, and on institutional pages.

Study status
Not yet recruiting.

Discussion
The protocol presented in this study demonstrates different modalities of a single intervention in aquatic physiotherapy considering low, medium, and high-intensities strategies and aims to evaluate their effects on biochemical behavior in pain, edema, and flexibility in patients with knee osteoarthritis.
The proposal of low-intensity intervention consists of movements that involve the upper and lower limbs and trunk and are highly recognized by their relaxation capacity, potentiated by the interactions between their practitioners and the properties of the liquid environment. 49This strategy has proven to be an effective method in the improvement of the symptoms and quality of life of patients diagnosed with Fibromyalgia, 50 reducing the risk of falls in older adults and producing an improvement in gait. 51Regarding knee osteoarthrosis, this treatment option demonstrated to be capable of positively interfering in the reduction of pain, stiffness, functionality, and the quality of life because of the thermal effects of the immersion and the interactions provided by the physical property of water associated with slow and relaxing movements. 43e aquatic physiotherapy intervention, when implemented, must take into consideration the body constitution, the speed of the movement, and the depth of the water as fundamental determinants to compose the intensity of the programmed exercise.Moving a segment in water requires more effort than moving oneself on the ground.The reduction of body weight experienced with the immersion also reduces the load on articulations affected by osteoarthritis and allows the performance of the closed-chain functional exercises that might otherwise be very difficult to perform on land.In the same way, the water turbulence might be used as a method to increase the resistance, and the percentage of body weight carried by the lower limbs can be decreased or increased directly proportional to the immersion depth. 22erefore, using the physical principles of the water becomes a great ally when aiming to treat individuals in this environment.For example, floating can reduce pain in patients with osteoarthritis since immersion is directly related to the reduction of body weight.In this situation, decreasing load means reducing articular pain, allowing a patient with osteoarthritis to perform exercises where the body weight variations can be constituted as an important therapeutic strategy for performing functional exercises, which is hard to adapt to the ground. 24e hydrostatic pressure is directly related to the immersion depth, which has a positive effect on venous blood return and reduction of edema. 26Water can retain the temperature and is an important thermal conductor capable of transferring thermal variations to the immersed body.The aquatic physiotherapy programs performed at temperatures ranging from 33.5-35.5°Callow heat transfer to the segments in contact with water and pain relief. 52dely used, aquatic exercises provide positive results in joint mobility and muscle strength because, with the immersion, the impulsion force provided by floating reduces the weight of body segments in a directly proportional way to the volume of displaced water, and this condition favors the movement of individuals with muscle weakness. 53enefits such as an improvement of functionality, muscle strength, and quality of life are observed in patients with osteoarthritis and other skeletal muscle disorders after aquatic physiotherapy. 52The implementation of treatment programs for knee osteoarthritis can also be planned from high-intensity exercises.This option of intervention is from adjustments in variables such as the increase in the period of training (weeks, months), frequency (days, weeks), session (minutes), or the amount of effort generated with the activity (heart rate, effort).This way, the composition of a program of high-intensity versus low-intensity must take into consideration the performance of the same program of exercises or physical activity in both groups, however, with different intensities. 47nsidering that this protocol aims to explore the effects of different programs of aquatic physiotherapy, to increment the work imposed during the performance of each one of the programs, the variables time, resistance, and movement speed will be proportionally modified.High-intensity training is known to bring benefits by contributing to functional performance and reducing the risk of falls for patients with knee osteoarthritis, and when performed in an aquatic environment, producing less load than when performed on the ground. 54Lower articular load and lower generation of pain are clearly observed in squat exercises performed in a swimming pool, which indicates the great therapeutic potential of aquatic physiotherapy for patients with this condition. 55e swimming pool walk is an important component for the improvement of the aerobic capacity of patients with osteoarthritis.This activity in the liquid environment requires speeds 30% lower than on the ground, while accelerating walks demand an increase of work of the cardiovascular system and require more strength for its execution.Walking with water at a depth corresponding to the xiphoid appendix reduces the articular overload, however, the increase in the speed can selectively recruit the hip extensor musculature, while walking backward reduces the patellofemoral compression, relieving compressive loads on this joint. 56[58] Nevertheless, although the improvement of functional variables in patients with osteoarthritis is widely unknown, the understanding of molecular mechanisms and their effects on articular cartilage are still scarcely explored.The effects of an aquatic exercise program on degenerative modifications of articular cartilage in an experimental animal model of knee osteoarthritis evidenced alterations in MMPs concentrations 26 and demonstrated that aquatic physiotherapy was able to produce more integrity and orientation of collagen fibers. 27In this context, the analysis of biochemical markers constitutes a relevant topic of study by evaluating the effects of different modalities of aquatic physiotherapy on the functional and biochemical behavior of patients with knee osteoarthritis and becomes more relevant when seeking understanding and answers obtained in different work intensity programs on articular cartilage.The expected results with one single session will be indicative for future longitudinal studies based on assertive intervention proposals regarding protocols with lower production of biochemical agents responsible for collagen degradation.
Finally, immersion-based physiotherapeutic treatment can prevent cartilage degeneration, inhibit inflammation, and prevent subchondral bone loss, as the presented evidence indicates that this modality can improve pain, stiffness, joint dysfunction, and muscle weakness in patients with knee osteoarthritis.Similarly, various treatment options are characterized by exercises of different intensities, such as aerobic exercises or strength training. 59Thus, the expected results in this protocol may support future longitudinal studies, contributing to assertive intervention proposals and constituting protocols aimed at reducing the production of biochemical agents responsible for collagen degradation.
The authors believe that this study, while providing important information, has some limitations.First, biomarkers considered important for understanding knee joint metabolism will be used.However, the inclusion of Cartilage Oligomeric Matrix Protein (COMP), a protein found in the extracellular matrix of cartilage, tendons, and other connective tissues, as well as other metalloproteinases and the analysis of inflammatory cytokines such as IL-1, IL-6, IL-8, and TNF-α, could offer a more detailed analysis of the acute effects of aquatic therapy on metabolism across all joint tissues.Second, the systemic response of the markers will be assessed.The authors are aware that analyzing intra-articular samples could provide information about this microenvironment; however, extracting intracavitary knee samples poses a significant challenge for implementing exercise in immersion, as sample collection involves invasive procedures in the joint cavity, which could contraindicate immersion immediately after collection.Third, interlaboratory variability and slight variations in existing study protocols can make it extremely difficult to compare results from different interventions to better understand the role of exercise characteristics and their impact on serum concentrations of soluble markers in this protocol.Fourth, the objective is to analyze the acute effects of different aquatic therapy protocols, but it is understood that future longitudinal studies, supported by the results from the implementation of this protocol, could provide better information on long-term interventions.

Conclusions
The positive effects of aquatic physiotherapy on functional variables in patients with knee osteoarthritis are widely known and studied, however, it is still necessary to deepen the understanding of this treatment modality on the interaction of biochemical mediators from the degenerative process of articular cartilage.Likewise, understanding how different exercise intensities affect these variables is essential, since the safe modulation of exercises intensities is one of the important parameters in the treatment of aquatic physiotherapy.In this way, the study of the effects of programs of variable intensities in a single session is the object of the present protocol, it may provide subsidies for new longitudinal studies aimed at the lower production of biochemical agents of articular degradation.

Domiziano Tarantino
1 University of Naples Federico II, Naples, Italy 2 Fondazione Don Carlo Gnocchi, Salerno, Italy Dear Authors, Even if the overall quality of your study protocol is good and with a solid scientific rationale, I am skeptical about such extremely short follow-up (24 hours).Please help me understand how the findings could be useful in clinical practice since osteoarthritis is a chronic conditions that needs therapeutic solutions in the long-term.
Kindly check the typing mistakes throughout the entire manuscript and some sentences need to be rephrased (such as "It is currently assumed that inflammatory processes osteoarthritis is strongly related to the genesis of osteoarthritis" or "instituting treatment through aquatic physiotherapy in patients with knee osteoarthritis means considering that", etc).
Some references are needed, such as at the end of the following sentence: "Previous studies in a systematic review demonstrated that aquatic physiotherapy positively affects clinical, functional, and quality of life variables in individuals with osteoarthritis."

Methods
Figure 1 is quite confusing.I would suggest to remake it.
No reference to what patients did or were making before the 15-day wash-out in terms of physiotherapy and/or therapeutic exercise.Thus, we believe that the relationship between the intensities and the Borg scale has already been adequately addressed.

Outcomes
Finally, the composition of Figure 1

Onu Ilie
Department of Biomedical Sciences, University of Medicine and Pharmacy "Grigore T. Popa", Iasi, Romania "Effects of a low, medium, and high-intensity aquatic physiotherapy protocol on functional and biochemical parameters in individuals with knee osteoarthritis: protocol for a crossover randomized controlled trial" It is a well-done scientific study but whether it can be indexed or not is conditional on the clarification of some issues: 1. 15 patients are too few to validate a study 2. GIFs in tables cannot be viewed 3. the study does not include limitations 4. To be replicated the study should have patients who fall into one KL classification (e.g.KL3 or KL 4).

a control group should be included in the study
After clarification of the issues mentioned above, the article can be indexed.
Is the rationale for, and objectives of, the study clearly described? of 15 individuals.We have included this information in the third paragraph of the Trial Design section.

GIFs in tables cannot be viewed:
The figures in all tables are static and, according to our access, are available on the website where the article is published ( https://f1000research.com/articles/12-1605/v2#referee-response-265791).They are also visible in the PDF version.We will report your observation to the publisher, who should find a solution to make them viewable directly on the website.This adjustment depends on the publisher and not the authors.

2.
The study does not include limitations: We appreciate the suggestion, and the limitations have been included in the text.

3.
To be replicated, the study should have patients who fall into one KL classification (e.g., KL3 or KL4): We have addressed this suggestion, and the change has been included in the new version of the article.

4.
A control group should be included in the study: We confirm that we have a control group.The last sentence of the Eligibility Criteria section states: "For control purposes, a reference group will be constituted with age matching and exclusion criteria identical to the experimental group."

Cesar Augusto Teixeira
Competing Interests: No competing interests were disclosed.

Version 1
Reviewer Report 06 March 2024 https://doi.org/10.5256/f1000research.153684.r246658 © 2024 Zhao Q.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Qian Zhao
Shanxi Provincial People's Hospital, Shanxi, China The submitted research delves into the effects of varying intensities of aquatic physiotherapy regimens on the functionality and biochemical parameters of patients with knee osteoarthritis, a topic of high significance.I commend the authors for initiating such a valuable endeavor.However, in my opinion, the authors should commence their introduction or theoretical framework by addressing the initial challenges present in the treatment of rheumatic joint disorders.They should then broaden the discussion to encompass the primary objectives of their research in osteoarthritis, including the authors' eventual determination of the importance and necessity of low, medium, and high-intensity aquatic physiotherapy.They should also reiterate commendation for this initiative, which has been discussed in recent years in relation to other rheumatic joint functional disorders and inflammatory diseases.
Another key point to highlight is that in terms of the study design, Figure 1 appears to lack the representation of a crossover design trial.Given the study's low, medium, and high-intensity aquatic physiotherapy combinations, there are six different sequences of administration, which are not apparent in the figure.Moreover, how was the final sample size of 15 determined?The paper seems to lack clarification on this matter.I suggest that the authors present the process of determining the required sample size, which would enhance the paper's content.Following the aforementioned recommendations, the discussion of the obtained data should be more comprehensive.
Is the rationale for, and objectives of, the study clearly described?Partly

Is the study design appropriate for the research question? Partly
Are sufficient details of the methods provided to allow replication by others?Yes

Are the datasets clearly presented in a useable and accessible format? Yes
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Rheumatism immunology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Figure 1 .
Figure 1.Flow of interventions and assessment parameters.

Table 1 .
Program A: low-intensity protocol.The individuals in the images are students of the Physiotherapy course who consented and authorized their image.ExercisePeriodization Description4 minutesAntero-posterior and latero-lateral walk.4 minutes, 2Â2 minutesHorizontal adduction and abduction of the shoulders alternately with rotation of the trunk to the left and right, aiming at transferring the weight in each of the lower limbs.Elevation of the lower limb forward (single leg support) during the horizontal shoulder abduction and adduction phase.Return to bipedal support during the horizontal shoulder abduction and adduction phase.

5 minutes
Immersion in the whirlpool bath Fifteen seconds rest between each exercise Total program time: approximately 45 minutes Table 2. Program B: medium-intensity protocol.The individuals in the images are students of the Physiotherapy course who consented and authorized their image.and latero-lateral walk.

2 Interventions
I would use T0, T1, T2 instead of moment 1 and moment When talking about high-intensity, please specify that you are referring to the Borg scale.Limitations of the study should be moved at the end of the Discussion.You should add the following references: 1) Tarantino D et al. (2023 [Ref -1]) 2) Tarantino D et al. (2023 [Ref -2]) References 1. Tarantino D, Theysmans T, Mottola R, Verbrugghe J: High-Intensity Training for Knee Osteoarthritis: A Narrative Review.Sports (Basel).2023; 11 (4).PubMed Abstract | Publisher Full Text 2. Tarantino D, Mottola R, Palermi S, Sirico F, et al.: Intra-Articular Collagen Injections for Osteoarthritis: A Narrative Review.Int J Environ Res Public Health.2023; 20 (5).PubMed Abstract | Publisher Full Text Is the rationale for, and objectives of, the study clearly described?Yes Is the study design appropriate for the research question?Yes Are sufficient details of the methods provided to allow replication by others?

Table 2 .
Continued was adapted from the article "CONSORT 2010 statement: extension to randomised crossover trials" (available at http://dx.doi.org/10.1136/bmj.l4378).However, we would be grateful if you could provide any other suggestions for flowcharts should you feel that it needs to be further modified.
https://doi.org/10.5256/f1000research.163915.r265791© 2024 Ilie O.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.